Combined antiretroviral therapy (cART) has dramatically changed the HIV epidemic, prolonging life and reducing morbidities. Nevertheless, a wide range of non-AIDS conditions continue to afflict people living with HIV, including cancers as well as cardiovascular, neurologic, kidney and bone diseases. These conditions are caused or influenced by inflammation states and immune activation and dysfunction that persist despite successful cART. In some cases, these conditions may be exacerbated by substance abuse, for example, cigarette smoking, which is more prevalent amongst HIV-infected individuals than in the general population. Extracellular vesicles (EV), including exosomes that bud into endosomal compartments, are membrane particles release by all known cell types that engage in intercellular communication. EV contribute to disease pathogenesis and are actively investigated, especially in cancers, as biomarkers and potential therapeutic platforms. Unfortunately, there is a relative dearth of information on the role of EV in HIV infection and substance abuse. To address this need in HIV and cigarette abuse, we have assembled an outstanding international team with combined strengths in HIV, EV, and immunology research, and technical portfolios ranging from EV isolation, nanoparticle characterization, and EV functional assays to bioinformatics, proteomics, lipidomics, and RNomics. Our investigators have identified a plasma signature of acute retroviral infection; characterized the roles of cellulr proteins in exosome and HIV release; unraveled the immunomodulatory function of EV; and confirmed effects of cigarettes on the immune system. Here, we will identify cellular proteins differentially involved in HIV and EV release (Aim 1), then use these findings and other methods to characterize omics changes to CD4+ T cell and macrophage exosomes and EV that occur with HIV infection or exposure to components of cigarette smoke (Aim 2). Functional studies will demonstrate how CD4+ T cell and macrophage EV released in HIV infection and nicotine abuse affect the qualities and function of cells that contribute to innate and adaptive immune responses (Aim 3). Finally, quantity, compositional quality, and function of circulating EV will be assessed in smoking and nonsmoking individuals who are diagnosed with HIV, both before and after they begin cART (Aim 4). The goal of these studies is to define the role played by exosomes/EVs in the promotion of pro-inflammatory states in HIV-1 infected individuals and smokers. Our findings will provide important insights into the pathogenesis of immune dysfunction/inflammation observed in these populations. We expect that our results will open pathways to new, likely EV-based, therapeutic interventions, helping to solve one of the major clinical challenges in HIV-1 medicine: the lack of treatments to limit immune activation and inflammation in HIV+ persons treated with cART.